The processes involved in the assembly of complex, multimolecular biological structures are poorly understood. This project seeks to use the striated muscle myofibril as a model system in which to explore these mechanisms. Extremely large myofibrillar proteins are hypothesized to play a key role in myofibril assembly. As a first step to studying the function of extremely large myofibrillar proteins using molecular genetic approaches, we are cloning, sequencing, and expressing portions of the mouse nebulin message. To date, we have characterized three clones encoding the central third of the 800 kDa nebulin protein. The three clones can be assembled into two distinct contiguous sequences that encode different isoforms of the protein. The translated sequence consists of a series of 245 residue repeats, in agreement with the published sequence of human nebulin. The translated mouse nebulin sequence is greater than 90% identical to the human sequence, with the exception of a 200 base pair region where the level of identity drops to 58%. We have constructed prokaryotic expression vectors carrying two of the isolated clones, and have succeeded in expressing 115,000 and 75,000 dalton fragments to high levels in E. coli. We are currently purifying the expressed nebulin fragments for use in in vitro experiments. In the course of this project, we had discovered a novel, muscle specific message with homology to nebulin. We have now cloned and sequenced over 90% of this protein. The sequence data predict a -115,000 dalton protein, with two nebulin-like 245 residue repeats near the C-terminus. We have produced an antibody against a synthetic peptide derived from the deduced amino acid sequence of this cDNA. In immunoblots, this antibody detects expressed fragments of the nebulin related protein in E. coli, and a single protein of the predicted size in mouse skeletal muscle. A slightly smaller band is detected in mouse cardiac muscle, indicating the presence of tissue specific isoforms of the nebulin related protein. These results also confirm our previous conclusion, based on northern blot experiments, that the nebulin related protein is expressed in both skeletal and cardiac muscle. We are currently purifying this novel protein for use in in vitro experiments, as well as using the specific antibody to localize it in the muscle cell.